The primary immunologic abnormality in HIV-infected patients with an infection in the active stage is the progressive depletion and functional impairment of T lymphocytes expressing the CD4 cell surface glycoprotein (Lane et al. (1985) Ann. Rev. Immunol. 3:477). Generally, T lymphocytes expressing the CD4 surface glycoprotein have a helper/inducer T cell phenotype (Reinherz et al. (1980) Cell 19:821), but such T cells can also have cytotoxic/suppressor activity (Thomas et al. (1981) J. Exp. Med. 154:459). It is believed that the loss of the helper/inducer functions in immunocompromised AIDS or ARC patients leads to the opportunistic infections and malignancies associated with AIDS.
Molecular studies of HIV infection of T cells have shown that HIV specifically and selectively infects T cells expressing CD4. It was also observed that CD4-specific monoclonal antibodies could block HIV infection and syncytia formation (Dalgeish et al. (1984) Nature 312:767; McDougal et al. (1985) J. Immunol. 135:3151). Maddon et al. (1986) Cell 47:333 showed that cells normally non-permissive for HIV infection which expressed a stable cDNA encoding CD4 became permissive for HIV infection. These results showed that CD4 was required for HIV infection. McDougal et al. (1986) Science 231:382, demonstrated complex formation between CD4 and gp120, the major HIV envelope glycoprotein.
cDNA encoding CD4 has been cloned and sequenced (Maddon et al. (1985) Cell 42:93). Sequence analysis shows an N-terminal signal peptide sequence, domains which exhibit homology to certain immunoglobulin variable-region domains, potential glycosylation sites at about 273 and about 303 in the amino acid sequence, a potential trans-membrane domain from about 375 to about 395, and a potential cytoplasmic domain extending through the C-terminus of the protein. Peterson and Seed (1988) Cell 54:65 performed site-directed mutagenesis of the CD4 protein to determine HIV binding sites, and correlated this information with epitopes recognized by CD4-specific monoclonal antibodies. Amino acid substitutions in the region of amino acids 45-47 of the protein appeared to destroy both HIV binding and syncytium formation.
Secreted, soluble forms of CD4 have been synthesized using truncated coding sequences. CD4 derivatives of about 370 amino acids have been produced; these are glycosylated when produced in appropriate host cells. Such molecules bind HIV gp120 effectively and can block HIV infection of susceptible cells (See, e.g., Smith et al. (1987) Science 238:1704; Fisher et al. (1988) Nature 331:76; and Hussey et al. (1988) Nature 331:78). Soluble truncated CD4 proteins as short as 113 amino acids, which are not glycosylated, can block HIV-mediated cell fusion (Chao et al. (1989) J. Biol. Chem. 264:5812). Thus it is clear that intact oligosaccharide side chains are not required for HIV binding or for cell fusion events.
The use of soluble forms of CD4 has been proposed for AIDS treatment or prophylaxis (See e.g., EP 0 385,909, published Sep. 5, 1990). Soluble forms of CD4 are known to have a short half-life in circulation in relation to certain serum proteins. Because the in vivo plasma half-life of soluble CD4 has been shown to be relatively short, various strategies have been employed to stabilize the protein against clearance. (See, e.g., WO 89/03222; WO 89/02922; WO 90/01035; and WO 90/05534). Conjugates have been prepared in which polyethylene glycol or other hydrophilic polymers are attached to the CD4 either via amino acid free functional groups or via sugar moieties in the oligosaccharide side chains of the glycosylated soluble CD4 protein. A second approach to stabilizing the CD4 protein in circulation has been to produce fusion proteins including a soluble CD4 portion and a portion from a protein of long circulating half-life, such as an immunoglobulin. Such fusions exhibited longer plasma half-lives in animal models than sCD4 without added domains.
Therapeutic proteins may be removed from circulation by a number of routes. For some pharmacologically active proteins, there are specific receptors which mediate removal from circulation. Proteins which are glycosylated may be cleared by lectin-like receptors in the liver, which exhibit specificity only for the carbohydrate portion of those molecules. Nonspecific clearance by the kidney of proteins and peptides (particularly nonglycosylated proteins and peptides) below about 50 kDa has also been documented. It has been noted that asialo-glycoproteins are cleared more quickly by liver than native glycoproteins or proteins lacking glycosylation (Bocci (1990) Advanced Drug Delivery Reviews 4:149). The sialic acid residues of erythropoietin appear to contribute to its stable circulation (Fukuda et al. (1989) Blood 73:84). In contrast, studies of tissue-type plasminogen activator (tPA) showed that the oligosaccharide sidechains were not the primary determinants for clearance from solution, but rather rapid clearance was dependent on the amino acid sequence within one or more domains of the molecule. The presence and type of glycosylation made a secondary, less significant contribution to clearance (Larsen et al. (1989) Blood 73:1842).
Mammalian glycoproteins often have N-acetylneuraminic acid (sialic acid) as the external (terminal) residue of the oligosaccharide chains which may be N-linked or O-linked (See, e.g., Osawa and Tsuji (1987) Ann. Rev. Biochem. 56:21).
Where the nature of the oligosaccharide is the primary determinant for clearance from circulation, generally glycoproteins with terminal sialic acid residues removed (asialoglycoproteins) are cleared more quickly than their intact counterparts. Circulating glycoproteins are exposed to sialidase(s) (or neuraminidase) which can remove terminal sialic acid residues. Typically the removal of the sialic acid exposes galactose residues, and these residues are recognized and bound by galactose-specific receptors in hepatocytes (reviewed in Ashwell and Harford (1982) Ann. Rev. Biochem. 51:531). Liver also contains other sugar-specific receptors which mediate removal of glycoproteins from circulation. Specificities of such receptors also include N-acetylglucosamine, mannose, fucose and phosphomannose. Glycoproteins cleared by the galactose receptors of hepatocytes undergo substantial degradation and then enter the bile; glycoproteins cleared by the mannose receptor of Kupffer cells enter the reticuloendothelial system (reviewed in Ashwell and Harford (1982) Ann. Rev. Biochem. 51:53).
Studies with asialo-ceruloplasmin and derivatives showed that asialo-ceruloplasmin in which galactose residues were oxidized by treatment with galactose oxidase and horseradish peroxidase and asialoagalacto-ceruloplasmin exhibited extended circulating half-lives as compared with asialo-ceruloplasmin (Morell et al. (1968) J. Biol. Chem. 243:155). Efficient removal by the galactose receptor appears to require at least two exposed galactose residues. In contrast, transferrin is a glycoprotein in which the sialylation state of the oligosaccharide is not key to rapid clearance from circulation (Morell et al. (1971) J. Biol. Chem. 246:1461).
From the foregoing cited examples of glycoproteins for which sialylation is the key determinant of clearance from circulation and those for which sialylation has no bearing on clearance or for which oligosaccharides play a relatively insignificant role in clearance, one may conclude that the fate of a particular glycoprotein in circulation and its apparent mechanism for clearance must be determined empirically. Similarly, strategies for prolonging the circulation of a particular glycoprotein must be evaluated on a case-by-case basis. The mechanism for clearance must be evaluated and the strategies for slowing or avoiding clearance must take into account maintenance of desired biological activity or function, potential toxicity, potential immunogenicity and cost.
A problem solved by the present invention is the prolongation of the circulating half-life of soluble CD4 derivatives, thus reducing the quantity of injected material and frequency of injection required for maintenance of therapeutically effective levels of circulating sCD4 for treatment or prophylaxis of HIV-infected individuals. The short in vivo plasma half-life of sCD4 is undesirable from the standpoint of the frequency and the amount of soluble CD4 protein which would be required in the prophylaxis or treatment of AIDS. The present invention provides means to prolong the circulating half-life of sCD4 with the most conservative but still effective change to the glycoprotein structure and with the substantial maintenance of gp120 binding activity.